Single-exon transcripts were oriented to positive strand or negative strand, depending on which strand resulted in higher read counts.
If an unspliced EST cluster overlaps with an predicted forward TSS peak in its first 84 nucleotides but not with an reverse TSS peak it is defined as a forward oriented transcript.
If a cluster overlaps a TSS peak on the opposite strand in its last 84 nucleotides but not with a forward TSS peak its defined as reverse oriented transcript.
Uracil-DNA-glycosylase (UDG) treatment preceded the PCR reaction to amplify exclusively the originally oriented transcripts.
These data comprise 250,035 correctly oriented transcripts or contigs that were kept for further analysis, as they appear to originate from viridiplantae or did not had any hits to nucleotide sequences in the NCBI databases (Additional file 2).
To discriminate between ESTs of antisense transcripts and wrongly oriented clones, only 3′ ESTs with a poly(T) signal and 5′ ESTs without a poly(T) signal were selected for this analysis.
In addition, we observed a correlation between intergene distance and plasticity (ρ = 0.19, p < 10−43, n = 5102), stronger when only transcripts with divergently oriented upstream partners are considered (ρ = 0.27, p < 10−57, n = 3271; Figure 5A).
Previous reports have shown that a large number of transcription-factor binding sites (TFBSs) for well-characterized transcription factors such as Sp1, c-Myc, p53 and Creb lie upstream to transcriptionally active intronic regions [ 41, 46], some of which generate transcripts that are oriented antisense relative to the known gene [ 46].
However, this finding was not consistent across all pairs since transcripts 3' of solo LTRs oriented sense and anti-sense were detected at similar relative levels.
Gene expression analysis further suggests that antisense transcripts generated from the oppositely oriented overlapping genes are differentially regulated and may have important regulatory functions in these mosquitoes.
